We are conducting studies to examine the role of human herpesviruses in CNS disease. In particular, we have focused on the ubiquitous virus HHV-6 (and HHV6B) that is associated with a variety of neurologic diseases including multiple sclerosis, encephalitis, epilepsy, and brain cancer. The generation of an animal model of HHV-6 infection would allow studies on the potential of this virus to cause neurologic disease. We initially inoculated marmosets with HHV-6A, HHV-6B, or vehicle control intravenously and followed disease development clinically, radiologically, and immunologically. Neurologic symptoms were observed in HHV-6A inoculated marmosets, predominantly characterized by sensory deficits. Symptoms were not observed in the HHV-6B or control inoculated marmosets. Many of the virus inoculated marmosets generated HHV-6-specific IgM and IgG, though most robustly in the HHV-6A inoculated group. By nested PCR, we detected little viral DNA in the periphery (plasma, PBMC, saliva). However, in a subset of marmosets, HHV-6 DNA was detected in CNS tissue, demonstrating that even in the absence of detectable virus in the periphery, virus may still be present in the CNS. We are also exploring the expression of the 18 kDa translocator protein (TSPO) that is strongly unregulated in CNS injury and thus of interest as a biomarker of neuroinflammation. Human exposure to HHV-6 occurs most likely through mucous membranes, and we have recently shown that nasal mucous is a reservoir for the virus. We therefore inoculated marmosets intranasally with HHV-6A, to examine a more physiologic route of exposure with the virus that was comparatively more immunogenic and neuropathogenic. In contrast to what we observed with the intravenous inoculations, marmosets inoculated intranasally with HHV-6A did not exhibit clinical symptoms, did not mount robust anti-HHV-6 antibody responses, and had a marked increase of detectable virus in the periphery (plasma, PBMC, saliva). These data are suggestive of an inverse correlation between antibody production and circulation of viral DNA in the periphery, and moreover, that the clinical symptoms observed in the intravenously inoculated marmosets may have been tied to the robust antibody responses. With this model we have generated a system in which to independently study the biology of the two HHV-6 species. This is an ongoing problem in the field, due to the high homology between HHV-6A and HHV-6B, the early exposure time to HHV-6B and the unknown time of exposure to HHV-6A. This also enables us to compare the development of neurologic disease with experimental autoimmune encephalomyelitis (EAE), a well-known established model for MS. EAE in the common marmoset develops cortical and white matter lesions with remarkable immunological and pathological similarity to those seen in MS. We have successfully established EAE in the marmoset, using human white matter homogenate as the auto-antigen to drive inflammatory CNS demyelination, with progressive or relapsing phenotypes. Using these methods of induction, we are able to implement clinical and MRI parameters that will enable us to test new disease modifying therapies. Using the 7T MRI, we now have a MRI protocol optimized to follow lesion development and quantitate lesion loads in affected marmosets. We are currently working to expand the MRI protocol in collaboration with Afonso Silva and Danny Reich in NINDS to obtain images of the spinal cord in the marmosets, to better track disease development and better correlate clinical changes with radiologic changes. We are investigating the radiological correlates of the tissue changes that accompany the formation of inflammatory, demyelinating brain lesions, the histopathological hallmarks of MS. We recently completed experiments to investigate the outcomes of EAE in marmosets previously inoculated with HHV-6, as viruses are considered a trigger of disease onset and progression in MS. Marmosets were intranasally inoculated with HHV-6A, HHV-6B or control material, and then several months later induced with white matter homogenate EAE. Viral inoculation was asymptomatic, and did not result in the induction of inflammatory cytokines in plasma. Moreover, during ex vivo PBMC stimulations with viral lysates, cytokine production was observed in only a subset of virus inoculated marmosets. Moreover, an HHV-6 specific antibody response was observed in a subset of the HHV-6B inoculated marmosets, but rarely in HHV-6A inoculated marmosets, consistent with our previous observations. Similar to what is observed in healthy adults, HHV-6B was preferentially detected in the saliva. All HHV-6B inoculated marmosets had detectable virus in saliva at multiple time points, but rarely in HHV-6A inoculated animals. Viral DNA was not observed in the PBMC of any HHV-6B inoculated marmosets. Interestingly, viral DNA was detected in the PBMC of the HHV-6A marmosets following EAE induction, reminiscent of findings of increased HHV-6A in MS patient PBMC. In one marmoset who was sacrificed following the last HHV-6B intranasal inoculation, viral antigen was detected sparsely in the brain parenchymain the absence of any other detectable neuropathologysuggesting that intranasal inoculation is sufficient for viral entry into the CNS. Following the induction of EAE, we noted significantly accelerated clinical disease in virus inoculated marmosets. This was accompanied by a greater induction of anti-myelin antibodies, and the earlier formation of brain lesions in these marmosets compared to controls. Examination of brain tissue from a marmoset inoculated with HHV-6B demonstrated the localization and upregulation of HHV-6 viral antigen within inflammatory nodules and perivascular cuffs of EAE lesions. This finding is significant in light of the fact that localization of HHV-6 viral nucleic acid and/or viral antigen around MS lesions has provided some of the most compelling data for its hypothesized role in MS pathogenesis. We are performing cytokine analyses and intracellular cytokine staining to examine the mechanism of accelerated disease in the virus inoculated marmosets. Post-EAE, the virus inoculated marmosets exhibited increased effector/memory CD8 cells, which significantly correlated with time post-EAE induction, while this was not observed in the control marmosets. As this CD8 subset is the highest producer of interferon gamma, this observation suggests a proinflammatory cytokine-mediated enhancement of EAE, wherein EAE induction served as a boost of CD8 effector function to a pool of virus-specific? cytotoxic T cells primed by the HHV-6 inoculations. Collectively, these observations support the hypothesis that asymptomatic intranasal viral acquisition accelerated and exacerbated subsequent CNS inflammatory disease through CNS infection and peripheral immune activation, both of which may promote blood brain barrier permeability and enhance inflammatory processes. These pre-clinical data further refine the association of HHV-6 with neuroinflammation and lend further rationale for prophylactic or therapeutic anti-viral interventions in patients affected by chronic or acute neuroinflammatory conditions. Recently, we have identified a naturally occurring marmoset herpesvirus, CalHV3, which shares homology with, and is hypothesized to be a primitive predecessor of EBV. We have observed a 60% frequency of detection in peripheral blood of NINDS colony marmosets, which is consistent with reports from other primate centers. This virus is also detectable at high levels in saliva, with patterns of fluctuation similar to EBV in human saliva. The presence of an endogenous marmoset EBV-like virus suggests its use as a preclinical model to explore the role of these agents in disorders such as MS in which EBV has long been suggested to play a role.
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